Saturday, December 13, 2014

In vino veritas: An elucidation of yeast-bacterial interactions in wine

By Abby Beltrame

Any
wine drinker will attest that there’s nothing better than a well-balanced,
flavorful glass of (my favorite, red) wine.Luscious flavors, slight acidity and tannins, a delicately smooth mouth feel,
and bountiful enticing aromas all contribute to a successful pour.Most wine drinkers also know, unfortunately,
the smell and taste of a spoiled vintage.How tragic!What you may not know
is that to achieve that perfect balance, or to prevent a spoiled disaster, a
subtle and complicated relationship must be perfectly managed.

The skins of these grapes are host to a myriad
of bacteriaand yeast species. [Author’s photo]

So what is it that
makes the composition of a great wine so complicated and the task of creating
such a wine so challenging? This is in large part due to that the flavors and
aromas of wine are the result of microbial metabolic products.The substrates for these metabolic processes are
the compounds found in grape must, a liquid concoction derived from the
pressing of grapes.Must consists not
only of grape juice but also the seeds, skins, and pulp.Found naturally within this mixture are
various microbes, a natural assortment of bacterial and yeast species1.The sensory-related components of the wine
are highly dependent upon the types of microorganisms present and both their cooperative
and antagonistic interactions.These
interactions depend on a number of microbial chemicals and byproducts,
including ethanol, lactic acid, glycerol, and inhibitory toxins.

It is important to
start at the most basic level- how is wine made?In essence, there are two main steps required
to make a palatable wine: a primary alcoholic fermentation and a secondary
malolactic fermentation.The primary
fermentation is most commonly carried out by the yeast Saccharomyces cerevisiae, during which the yeast converts sugars
into alcohol.Bacteria are the key
players in the secondary fermentation. Though not actually a fermentation, but
called as such, malolactic fermentation (MLF) is the enzymatic conversion of
malic acid to lactic acid1. MLF is accomplished by lactic acid bacteria
(LAB), primarily by the bacterium Oenococcus
oeni (formerly known as Leuconostoc
oenos)1.

Essential to
composing a tasty wine is balancing these yeast-bacteria interactions and the metabolic
compounds produced by each organism. Ethanol
produced by yeast contributes to the pleasing, psychotropic effect found in any
alcoholic beverage2. Glycerol,
a simple alcohol, is a secondary product of yeast fermentation and is known to
contribute to the texture of wine, lending a full-bodied smoothness, while also
augmenting the wine’s flavor profile3. Lactic acid production by LAB is equally
important not only because it deacidifies and stabilizes the wine, but also
because lactic acid imparts desired flavors and aromas that enhance the wine
quality4. Even so, the acidic pH, high ethanol content, and low
availability of nutrients that are byproducts of the primary fermentation by S. cerevisiae result in conditions that
do not readily support LAB growth1.On the other hand, LAB can carry out additional non-MLF metabolic
reactions that can result in off-putting aromas, undesirable flavors, and
overall spoilage of the wine1.It is not surprising then, that the complex interactions between S. cerevisiae and LAB involve both
inhibitory and stimulatory reactions by both the yeast and bacteria.

The
most commonly reported relationship between the two organisms is yeast inhibition
of bacteria. Early scientific studies found that in grape juice with mixed
cultures of S. cerevisiae and O. oeni, yeast has a shorter lag phase
and grows at faster rate than bacteria5. While ethanol production by yeast has long
been known to inhibit the growth of LAB, high ethanol concentration does not
seem to have a substantial effect on MLF activity alone4. Sulfur
dioxide (SO2) produced
by yeast can also slow bacterial growth, reducing the risk of spoilage, yet
also inhibits MLF4.Other
yeast metabolites, such as medium-chain fatty acids, have been shown to reduce
bacterial growth and MLF ability4.As a compounding effect, this inhibition is greatly increased at acidic
pH.

To
augment this inhibition, certain strains of S.
cerevisiae have been shown to encode genes for killer toxins that act
specifically on competitive microorganisms, including LAB6.These toxins, known as killer factors, are
cell-surface proteins that act at receptors on the bacterial cell wall.Strains of S. cerevisiae have been shown to produce three of these toxins
while under winemaking conditions, and while they can efficiently inhibit
bacteria, the toxins also restrain other yeast species6. Traditionally, winemakers add additional SO2 to wine to halt undesired microbial
growth and prevent spoilage.The use of
killer toxin-producing yeast strains is promising because they can act on their
own to control microbial overgrowth and replace or reduce the amount of SO2
used in winemaking6.

While it is commonly thought that yeast
overpower bacteria in the winemaking process, LAB can in turn inhibit yeast.In lower alcohol conditions, rapidly
proliferating bacteria cause a faster death rate of the yeast5.Interestingly, at a lower Brix (a winemaker’s
measure of sugar content), although LAB declines more in lag phase when
co-inoculated with yeast in grape juice, the bacteria has a faster logarithmic
growth rate.Thus at lower sugar
concentrations, the bacteria ultimately reaches a higher clonal population,
resulting in more rapid malolactic acid conversion5.

Another bacterial
effect is that many types of LAB metabolically degrade glycerol, resulting in
disagreeable organoleptic properties3.Some LAB, such as O. oeni, can produce glycerol under specific conditions,
particularly when nutrients are limited3. This can be used to the benefit of the
winemaker, by allowing conditions that support glycerol production while
minimizing bacterial overproliferation and spoilage of the wine.

In addition to the
inhibitory effects of wine yeast towards bacterial co-inhabitants, some
evidence suggests that stimulatory effects also persist between the two groups4.Yeast autolysis results in the release of
many nitrogenous compounds, including whole proteins, small peptides and individual
amino acids.Some of these nitrogen
isolates derived from wine have been shown to support LAB proliferation4.Taken together with the strong inhibitory
interactions, this supportive effect on the bacteria suggests a mechanism of
high bacterial inhibition while the yeast is active during the primary
fermentation phase, but then a transition to conditions that support bacterial
MLF once the yeast population reaches stationary phase and subsequently
degrades.

As
a novice winemaker, or just as a wine-lover, it is beneficial to understand how
to take advantage of these interactions in order to produce a superior wine. A
recent study by Ale et al. focused on the interactions between S. cerevisiae, O. oeni, and another common wine yeast Kloeckera apiculata3.After much experimentation using different concentrations of each
organism and playing with inoculation times, the authors of the study propose a
model inoculation scheme.Their proposal
promotes ideal alcoholic fermentations by yeast, while encouraging glycerol
production and MLF by the bacteria.Ale
et al. recommend a simultaneous inoculation of 106 CFUs/mL of S. cerevisiae with the same amount of K. apiculata, followed by a sequential
inoculation of 106 CFUs/mL O.
oeni.By heeding these
recommendations, any winemaker can hopefully increase the chances of concocting
a brilliantly balanced wine.

Pressing the grapes extracts the sugars for
yeast to feed upon,while also brings the many types of yeast and bacteria inclose contact, encouraging a suite of interactions. [Author’s photo]

It
is important to note that a whole chorus of microbes may be present on the
skins of grapes and, as such, there are likely many more interactions that
exist.Also, these interactions are not
isolated and often simultaneous, presenting further challenges in their study.While lab investigations into these relationships
often contain only S. cerevisiae and
LAB in grape juice-containing media, the many additional species of microbes
residing in the vineyard and winery contribute in their own ways to the success
or spoilage of a wine.More research to
elucidate these relationships will allow winemakers across the globe to better
harness the microbial processes that contribute to the delicious beverage that
many of us love. Even so, the key relationships discussed here contribute
greatly to the outcome of a wine, and I look forward to implementing them in my
own winemaking practices, as should you! And with that, I will end this with a
toast: Here’s to never sipping a spoiled wine again. Cheers!

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